WO2003023057A2 - Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells - Google Patents

Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells Download PDF

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Publication number
WO2003023057A2
WO2003023057A2 PCT/EP2002/005489 EP0205489W WO03023057A2 WO 2003023057 A2 WO2003023057 A2 WO 2003023057A2 EP 0205489 W EP0205489 W EP 0205489W WO 03023057 A2 WO03023057 A2 WO 03023057A2
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Prior art keywords
cells
characterized
portions
antibodies
mrna
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PCT/EP2002/005489
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German (de)
French (fr)
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WO2003023057A3 (en
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Winfried Albert
Pia Steffens
Alf-Andreas Krehan
Stefanie WASCHÜTZA
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Adnagen Ag
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Priority to DE10143776.5 priority Critical
Priority to DE10143691 priority
Priority to DE2001143775 priority patent/DE10143775A1/en
Priority to DE10143699 priority
Priority to DE10143691.2 priority
Priority to DE2001143776 priority patent/DE10143776A1/en
Priority to DE10143775.7 priority
Priority to DE10143699.8 priority
Priority to EPPCT/EP01/13606 priority
Priority to PCT/EP2001/013606 priority patent/WO2003044224A1/en
Application filed by Adnagen Ag filed Critical Adnagen Ag
Priority claimed from ES02772273T external-priority patent/ES2284927T3/en
Priority claimed from US10/488,828 external-priority patent/US20050014208A1/en
Priority claimed from EP20020774579 external-priority patent/EP1409746A2/en
Priority claimed from AT02772273T external-priority patent/AT359377T/en
Publication of WO2003023057A2 publication Critical patent/WO2003023057A2/en
Publication of WO2003023057A3 publication Critical patent/WO2003023057A3/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966Animal cells

Abstract

The invention relates to a method for selecting and/or qualitative and/or quantitative detection of predetermined biological cells from, for instance, a sample or in a sample containing biological cells, said sample being mixed with a predetermined combination of at least two antibodies and/or antibody derivatives which preferably bind via the binding sites thereof to various epitopes of the cells which are to be selected or detected, and/or with at least one biospecific antibody and/or antibody derivative which preferably bind(s) via the binding sites thereof to various epitopes of the cells which are to be selected or detected. The cells which are marked with at least one of the antibodies and/or antibody derivatives are separated from the sample and the separated cells are monitored by means of a predetermined combination of at least two molecular-biological analytical reagents which preferably react with at least one component of the cells which are to be selected or detected.

Description

Method and diagnostic kit for selectioning and / or for qualitative and / or quantitative

Detecting cells

The present invention relates to a method and a diagnostic kit for selectioning and / or for the qualitative and / or quantitative detection of cells in a sample. Such detection methods and diagnostic kits are particularly needed in the diagnosis or treatment control of tumor diseases. Because under the Krebsvor- or post-natal care, it is of great importance malignant tumors or re- zidive malignant tumors by the appearance of metastatic tumor cells in the blood sierender to demonstrate at an early stage. The present method and the present diagnostic kit are not only used here but can fundamentally for the detection and identification of rare cells in biological cells containing samples are used. This can be done for example, to detect föta- len cells in maternal blood or also for the detection of stem cells.

Testicular cancer is responsible for less than 2% of all malignant neoplasms of tumors in men.

However, it is at 20-30% of all cancers among men 40 years to testicular cancer. The annual number of new cases, for example in the Federal Republic of Germany is approximately 3,600, o- die of testicular cancer in 240 men. The highest incidence is found here between 25 and 40 years. By the progress in oncological therapy over 90% of all concerned in the long term can be cured today. advise the high survival reasons thereby in the pronounced efficacy of cisplatin-based chemotherapy.

Breast cancer is the most common diagnosis when a tumor disease in women is found (26.4% al 1er new cases). Despite massive efforts being expended in early detection, treatment and follow-up, this disease still ranks first cancer-related cause of death in women. strieländern the disease numbers in western INDU take in recent years despite increased efforts to early to rise. The problem is the high rate of metastasis after primary treatment, which already after 1-3 years results in the majority of cases the death of the patient. The main reason for this is the spread of tumor cells in early stages of tumor development. In addition to the first identification of a breast cancer is therefore in particular, the earliest possible detection of metastatic cells for successful treatment of decisive importance. Similarly, a definite negative evidence, clinical stage I can be helpful when deciding whether the patient therapy with chemotherapy or surgery must be loaded.

In colorectal tumor progression primary tumor after resection can be attributed primarily to residual tumor cells. These cells are detached pre- or intraoperatively from the primary tumor and given the opportunity to dispersal in the whole organism.

In addition to the first identifier of colorectal cancer therefore is particularly the earliest possible detection of metastatic cells for successful treatment of critical importance.

Here it must be decided in clinical stage 1 of the disease, whether the patient has to be loaded with chemotherapy and / or with surgery to achieve a permanent cure success. A large number of patients is thereby treated chemotherapeutic rapeutisch although no proven evidence of metastasis is present. In the concepts that are based on a pure monitoring, however, it comes in 25% of cases to recur with a fatal outcome in some cases.

In the currently used methods of investigation so-called tumor markers at the protein level (immunological or enzymatic) quantitatively averages in the blood or other body fluids ER in cancer patients. However, these detection methods have only limited usefulness, since elevated tumor marker values ​​in body fluids by non-tumorous diseases, such as inflammations of the gastrointestinal tract, liver cirrhosis, viral infections or heavy smoking for tumor diagnosis and treatment monitoring / follow-up of tumors kön- caused NEN.

Molecular genetic methods appear here for the detection of tumor cells in peripheral blood helpless rich, since the transfer of tumor cells can stand the venous blood at the beginning of the metastasis process.

EP 0520794 Bl discloses such a procedural reindeer, wherein metastases in body tissues or

Liquids are detected. In this case nucleic acids are detected, for example by means of amplification by polymerase chain reaction. The method is now based crucial that the detected nucleic is acid sequence expressed in cells of the tissue of origin of a tumor, ie in tumor cells and marker depends also in the healthy cells of the origin tissue. A further condition is that this sequence does not normally, is in those cells expri- mized whose tissue is examined. Thus, if a corresponding sequence found in the sample under investigation, it must result from protracted, ie meta- stasierenden cells of a non-local tumor. Thus, this method is ultimately based on the detection of cells that should not occur in the blood of healthy people.

The overall conclusion is that the diagnostic methods currently used are not accurate when it comes to assessing the malignant potency of residual tumors after chemotherapy performed in the meta stasierenden stages. It is therefore necessary to continue to find evidence of an occult or residual metastasize that permit timely integration into the diverse primary curative therapeutic options. Significant problem, it is here that the cells to be detected, for example, tumor cells in the peripheral blood, only occur in very small numbers.

Starting from this prior art, it is an object of the present invention to provide a method and a diagnostic kit is available, with the rare simple, safe and repeatable manner, biological cells in a biological cells corresponds holding sample with high sensitivity and specificity can be selected and / or detected.

This object is achieved by the method according to claim 1 and by the diagnostic kit according to claim 29th Advantageous further developments of the inventive method and the diagnostic kit of the invention are given in the respective dependent claims.

Critical in the novel process it is now that the first to selektionierenden or cells to be detected are kiert by means of a combination of antibodies or antibody derivatives or a bispecific antibody or antibody derivative MAR. This makes it possible, in particular to highlight the cells sought to separate and enrich it. This means that in a first step is a combined immunological detection or selectioning. any type of modified antibody or antibody fragment is understood derivative to antibody in this application that has a binding site, for example once-chain antibodies, antibody fragments such as Fab fragments or recombinant antibodies. Always antibodies and / or antibody derivatives are hereinafter referred to when speaking of "antibodies *, respectively. at least one marker ER- In a second step, on a molecular basis of a predefined combination of detection reagents bordered, which is specific for the sought-cells or be found preferentially in this, so that here again specifically the cells are to be selected. So we have here a combined molecular detection. The basic idea of ​​the present invention is thus to combine a detection via a combination of immunological parameters with evidence of a combination of molecular biological parameters. Surprisingly arising thereby quite outstanding detection results, which is advanced into detection areas all known techniques from the prior art were not accessible before. So concentrations sought cells in blood can be detected down to two cells per 5 milliliters. Such specificity and Senisitivität is not achieved so far in the prior art.

Eukaryotic cells carry a variety of different molecules at their cell surface. According to the origin and function of each cell, the combination of the ex- primierten surface molecules differs, so that cell-type specific patterns. To detect this cell type-specific patterns antibodies are used. Antibodies bind with high specificity to their antigen, here to selected surface molecules. This property is used by means of specific antibody binding to identify cells based on their cell-type specific pattern and distinguished from each other. So different, for example the expression of special surface proteins of tumor cells from non-transformed cells of this cell type.

The detection of the marker is preceded by a selection of the target cells via the binding of different antibodies to the cells sought. For the expression of specific surface proteins of different cells of one type of cells of another type. For example, the expression of special surface Port A tumor cells from non-transformative-programmed cells is different this cell type.

Since the special pattern of surface antigens is different, for example in tumor cells and the blood cell typical patterns, tumor cells can be distinguished in the blood. In order to identify tumor cells, antibodies that specifically recognize these special surface proteins are used as tools. The specific Antikörperbindunq is made usable for various analysis and separation methods.

Because of the intense bond of specially selektionierten for immunoglobulins also a separation of the detected cells in addition to the detection of cell surface epitopes on their unrecognized possible.

Various separation principles are possible:

1. Separation Principle Based on liquid phase; eg flow cytometry:

For the flow cytometric analysis are anti-body coupled to fluorescent dyes. Scattered cells are bypassed in a constant liquid stream individually to a light source (laser). Upon illumination of the cells bound to the antibodies, fluorescent dyes are excited and emit light of certain wavelengths. The emitted light is detected and the measured signal stored digitized. The light signal individual cells can be assigned. The Antikör- per-labeled cell is detected so and can now be separated from other cells. To separate the cells are isolated in small drops. After detection of the antibody-labeled cell of the corresponding droplet is directed into a collecting container. Such enrichment can occur for example by FACS flow cytometry. Here, for example enriched cells with fluorescently labeled antibodies are incubated monoklonaren against tumor-specific surface proteins. The labeled cells are washed twice with PBS and subsequently 10 7 cells are resuspended in 1 ml PBS. For the isolation of the tumor cells a FACS Van days SE flow cytometer (Becton Dickinson) is used. would take over the Cell Quest program carried out of data traffic, instrument control and data analysis. The sorted cells are incubated in a 1.5 ml reaction vessel (filled with 1 ml PBS) was transferred. The RNA can then be isolated as described later.

2. Separation Principle Based on solid phase; for example, magnetic separation:

For the magnetic separation, antibodies are coupled to pseudo-magnetic particles. After introduction of the pseudo-magnetic particles in a magnetic field, the particles move in the magnetic field. During the movement in this magnetic field, cells to which these antibodies are bound coupled entrained and separated from other cells.

consequently, antibodies to the cell detection by means of magnetic particles to pseudo-magnetic particles which have a defined number of chemically activated sites on the surface thereof coupled. Coupling methods, for example, from James P. Gosling, solid-phase Concepts and Design, in: RF

Masseyeff, WH Albert NA Staines (eds), Methods of Immunological Analysis, Vol. 1, VCH Verlagsgesellschaft mbH, Weinheim, pp. Known 507-529. On the specificity of the antibody, the specificity of the separation is determined. Blood sample containing a target cell is combined with antibody-coupled magnetic particles; then particles and blood are relatively moved "over-rotating head" for example, in a closed container befindli- more samples or by movement of the particles due to changing magnetic fields. Those target cells that are recognized by a solid-phase-bound antibody and thus firmly bonded, follow the movement of the particles. This makes it possible, when applying a magnetic field, the particles with the bound cells from the blood extract (for example, on the wall of the separation vessel). This can be exchanged for other solutions in this way target cell-depleted blood, the buffy cells magnetic particles to shutdown / removal of the

remain magnetic field on site and are available for further applications.

Alternatively to the illustrated separation principles, any other separation principles of the prior art, which rely on the labeling of cells with antibodies are used.

According to the invention are advantageously used specific antibody mixtures for the detection of tumor cells. For example, is suitable for the detection of tumor cells in the blood of a combination of the antibody MOC-31 and Ber-EP4.

Table 1: antibody mixture

Figure imgf000012_0001

By means of the antibody mixture in the foregoing Table 1 are preferred, but detected tumor cells with high specificity. This is based on the preferential expression of particular surface proteins which distinguish cancer cells from other cells.

Such antibodies mixtures exhibit compared to the separately antibodies used in the cell detection and cell separation regardless of the method used, an increased sensitivity.

The present invention is further based essential that not be detected as immunological or enzymatic shearing plane cell markers in the blood of patients, but that a molecular biological markers, such as mRNA (messenger ribonucleic acid) of desired cells in a sample such as a blood sample, is detected.

Since individual markers are differentially expressed in therapy-dependent manner, is advantageously studied a combination of tumor markers in order to detect all circulating blood tumor cells. In this way, tumor cells can also are recognizable when the expression of a particular marker is relatively low in a patient or in a disease state, which could otherwise lead to a supposedly negative result. However, the use of markers encounters mostly so on limits because kleäre mononuclear blood cells a background expression ( "illegitimate transcription *) which hamper a precise analysis.

As markers, for example for tumors, the expression of the genes listed in Table 2 is detected. The detection can be carried out with one another for one or two marker, or for any number of these tumor markers in combination. The kit of the invention may therefore two Oligonukleotidpaa- re for one, two or any selection or to contain all of the tumor markers.

table 2

Figure imgf000014_0001

This means that all the cases properly be neglected, for example, where due to other diseases also be tumor markers expri- ated and only get as protein in the bloodstream. consequently, it only be detected cells due to the first immunologic selection step that are even on the one hand in the blood sample and the other expressing the or the respective tumor markers. It is therefore to tumor cells derived from their original tumor tissue and were abducted in the blood of patients. Since, in the blood is not the mRNA of the studied marker is not normally expressed in a tumor ill patients, itself shows a direct correlation between the occurrence of the associated mRNA and a metastasis at an early stage in the process approximately metastasis.

Here vorteilhaf enough, not only the mRNA of a single tumor marker is detected but tested a combination of markers. This makes it possible to detect cancers through its metastasizing in the blood cells. This means that in the case of testicular tumors in both seminöse and nichtse- minöse testicular cancers or tumors also mixed with portions of a seminoma and therefore 90-95% of all malignant tumors of the testis, namely Moren all Keimzelltu- detected.

Therefore, a combination of at least two of the following markers the invention provides for the detection of testicular tumor cells:

- GA733.2

- GCAP / PLAP

- HMGI-C

- GRPR,

For the detection of breast cancer cells a combination of at least two tumor markers of the following groups according to the invention mormarkern proposed:

a) EGFR, CEA, stanniocalcin, MAGE 3, CK20, claudin 7, Her-2 / neu, MUC1 and GA 733.2; b) CK20, MAGE 3 and MUC1 c) Her-2 / neu and Claudin7 and d) EGFR, CEA and stanniocalcin

For detecting colorectal cancer cells a combination of at least two tumor markers of the following groups According to the invention mormarkern proposed:

a) CK20, EGFR, GA 733.2, CEA and stanniocalcin b) CK20, EGFR, CEA and stanniocalcin and c) EGFR, CEA and GA 733.2

Some examples are given, which show that the inventive procedural ren a detection sensitivity is achieved so far known about everything in the art goes far. Figures 1 to 10 show results of different experimental protocols.

Common for all examples is the basic procedure, which comprises a first step with an immunological enrichment of target cells and a second step of detection of mRNA markers in the immunologically enriched cells. In the fol- constricting the procedures are described in general terms, unless they are identical for all examples.

1. Immunological enrichment of target cells from peripheral blood

First, a peripheral blood sample was taken and this is added a defined number of target cells, for example, 2, 10, 100 cells of a certain type of tumor.

Furthermore, antibodies were pelt gekop- to magnetic particles. The antibodies represented below in Table 3 were used as antibodies.

table 3

Figure imgf000017_0001

The magnetic particles were thereby (Dynal Cellection ™ Pan Mouse IgG Kit) with a particle concentration of 4 x 10 8 beads / ml. The ratios between the antibody concentration and the antibodies coupled thereto are set forth in Table 4 below.

table 4

Figure imgf000017_0002

The prepared magnetic particles were added depending on the experimental approach and detection system of the blood. The corresponding additional antibody-coupled magnetic particles per ml of blood at a starting concentration of 4 x 10 8 beads / ml particles is wiedergegebe in Table 5 below.

table 5

Figure imgf000018_0001

After 2 hours of incubation in an overhead shaker the magnetic particles, optionally present as cell antibody-magnetic particle complexes were washed means of a magnetic (MPC ® -S, company Dy- nal) 3 times with PBS (phosphate buffer saline) and the adherent cells are then treated according to the method described in the following RNA Isolierungsproto- Kolls.

As an alternative to separation by means of magnetic particles to an immunological separation by means of flow cytometry (fluorescence-associated cell sorting, FACS) offers.

Here is a first relative enrichment of tumor cells is achieved by depletion of the erythrocytes. For this purpose, whole blood is mixed (with EDTA) with a hypotonic erythrocytes lysis buffer and incubated for 30 minutes at room temperature. The remaining nucleated cells are centrifuged and resuspended in PBS / BSA. The cells thus obtained are then incubated with antibodies which are labeled with a fluorophore. The target cells are fluorescently labeled by binding to an antibody were then separated on FACS.

Alternatively, an enrichment offers by dense tegradienten centrifugation. By such a centrifugation density gradient with different cells of different average volume density are separated. Mononuclear blood cells by means of a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden), separated and then washed twice with PBS / 1% FCS. Subsequently, a solid phase coupled enrichment (for example via magnetic particles) and a liquid-based separation (FACS) of the target cells as described above ben.

2. mRNA isolation

First, an isolation of the total RNA of the separated cells is carried out as described above. This is done with the QIAamp RNA Blood Mini Kit (Qiagen Company gen, Hilden) according to the manufacturer's instructions therein, wherein the lysis buffer was added directly to the bound to the magnetic particles cells. By an additional loan DNS digestion on the column contamination is avoided by genomic DNA. This DNA digestion takes place with the RNase-Free DNase Set, Qiagen, Hilden.

Alternatively, a mRNA isolation, eg. Done as -coupled means 01igo (dT) magnetic particles Dynabeads ® mRNA Direct ™ Micro Kit (Dynal)). Also, this isolation is according to the indicated in the kit manufacturer's instructions.

As a further alternative for RNA isolation, the isolated cells are lysed by the addition of Trizol reagent (Gibco BRL, NY, USA) and homogenized using a pipette. After subsequent chloroform extraction, the RNA-containing aqueous layer is precipitated in isopropanol at -80 ° C. After two washes and centrifugation in 80% ethanol, the pellet is air dried and then resuspended in RNase-free water. This work-up step also takes place kollen by conventional prototypes.

3. Reverse Transcription

The isolation of RNA a reverse transcription follows, in which the mRNA is transcribed into cDNA.

For this purpose, the RNA in a corresponding volume of water according to the reaction batch in Table 6 together with oligo (dT) 15 primers (Promega, Mannheim) is denatured for 5 minutes at 65 ° C and then incubated on ice. Table 6: Components of the cDNA synthesis

The cDNA synthesis is carried out in a 20 reaction mixture μl-

Figure imgf000021_0001

The cDNA synthesis was carried out at 37 ° C for one hour with subsequent inactivation of the reverse transcriptase by heating for 5 minutes at 95 ° C and subsequent cooling on ice. For this, a script Sensi reverse transcriptase kit, Qiagen, Hilden was used by the protocols indicated there.

In case of using oligo (dT) -coupled magnetic particles already for the isolation of mRNA subsequently remains the addition of oligo (dT) primers, that is, the oligo (dT) linker serves as a primer for reverse transcription, in which case the magnetic particles in the remain approach.

PCR

Subsequent to the transcription of the mRNA into cDNA is carried out a polymerase chain reaction (PCR) with .beta.-actin as an internal control.

The Oligonu- listed in Table 7 were used kleotide as PCR primers for amplification of cDNA corresponding to various marker genes, as indicated in the first column.

Table 7: List of PCR primers

Figure imgf000023_0001

Table 7 lists in the first column, the indication of the detected Tu ormarkers, each two olive gonukleotide (sense and antisense) is given as a primer pair. The length which is generated by the primer indicated in column two of the PCR product, is indicated in column three. The PCR was performed with the specified in Table 8 approach.

Table 8: PCR approach

The PCR synthesis was carried out in a 50 ul Rekationsansatz

Figure imgf000024_0001

(* Containing 15 mM MgCl 2; ** HotStarTaq ™ polymerase THEN; Qiagen, Hilden) (*** The addition of 10 ul Q-Solution (Qiagen, Hilden) is only for the detection of GCAP / PLAP necessary)

Table 9 provides a listing of the specific primary merkombination and primer concentrations as the final concentration in the PCR mixture to. In the following examples, breast cancer, colon cancer and testicular cancer is in each case an exemplary manner shows a multiplexing combination of these primers for the different types of tumors, such as it is given in Table 9 below. Table 9: List of the specific combinations Primerkom- and primer concentration (final concentration in the PCR mixture)

Figure imgf000025_0001

The PCR conditions (number of cycles, cycles guide etc.) are given in Tables 10 and 11. FIG. Table 10: PCR conditions

Figure imgf000026_0001

Table 11: temperature multiplex specific Annealingtempe- and number of cycles

Figure imgf000026_0002

(Thermocycler:; Biozym PCT 200)

The amplified products of the cDNA so produced were electrophoretically separated by means of a Bioanalyzer 2100 (Agilent). For this purpose, 1 ul of the PCR product was separated in the Bioanalyzer on a DNA chip (500) and the separation result documented electronically. In this manner, the figures 1-10 were generated.

Alternatively, 25 .mu.l of the PCR product are separated on a 2.5% agarose gel and stained with ethidium bromide, the DNA bands. ER documentation such as follows using the DUO Store Systems Intas.

Alternatively, a fragment may further analysis examples play by means of an ABI 310 Genetic Analyzer Pris (from PE Applied Biosystems, Weiterstadt) are performed. For this purpose, each 1 ul of the PCR product is then used in the dilution of 1:50. In this case, fluorescence-labeled primers are used.

Figure 1 now shows the result of an inventive method for the detection of breast cancer cells in the blood. For this blood was added by healthy a defined amount of tumor cells in a breast cancer cell. The added number of cells was 10 cells (10 Z) respectively. 100 cells (100 Z) per milliliter of blood. Figure 1A and 1B respectively show now electrophoretic separations retical, the individual bands are labeled here and below by the same terms. The term conductor designates Cali bratoren of 50-600bp length, with RT-Ko is a control called that did not contain any mRNA with PCR-Ko is a control measurement referred to contained no cDNA before PCR. By "blood * is the blood sample without inoculated tumor cells, with 10 Z denotes the blood sample with 100 inoculated tumor cells per milliliter or per 5 milliliters of the blood sample with 10 inoculated tumor cells per milliliter, and with 100 Z. By "cell line * a control measurement with a large number of cells of the tumor cell in the sample is indicated.

In Figure 1 results are shown when the lesson Se in the first step with only one, two or all three of the following antibodies HMPV.2, GP1.4 and Ber-Ep was performed. 4 It is immediately apparent that, when using only an antibody of the detection of the tumor marker CA 15.3 (MUCL) is small. The best results are obtained when two of the antibodies, HMPV.2 and Ber-EP 4 or GP1.4 and Ber-Ep 4 are used for detection. is already the combination of all three antibodies, as can be seen in the intensity of the bands for the tumor marker CA 15.3, more effective ver. This demonstrates that with a suitable choice of a certain number of specific antibodies, a significantly improved result in the detection of tumor cells possible is. In particular, also shows that the simple conclusion that with the use of multiple antibodies, the sensitivity would rise forcibly, is not possible. The opposite is possibly the case as a non-specific reaction with an increasing number of antibodies is more likely. It is therefore processing of particular importance to experimentally determine a suitable combination of antibodies.

Figure 2 shows results of detection methods in which no pre-selection using antibody labeling and in Figure 2B, a pre-selection was carried out using antibody labeling in Figure 2A. In Figure 2, thereby combinations of the antibodies HMPV.2, Ber-EP 4 and GP1.4 have been determined as a combination of two or all three antibodies. At the same time a multiplex determination of a total of four markers, namely GA 733.2, CA 15.3, Her 2 / neu and claudin 7 was carried out. Here too, 100 and 1000 tumor cells of a breast cancer cell line were 10 again inoculated in blood and subsequently detected. Here, that a background expression of mRNA for some markers (GA 733.2, 15.3 and CA Her 2 / neu) is detected without antibody selectioning shows. Such background can avoid the mRNA to be examined cells when using any of the antibody combinations shown in Figure 2B for preselection. Interestingly in Figure 2B, in turn, that the use of three antibodies to the use of two antibodies, eg. is as GP 1.4 with Ber-Ep 4 is not necessarily superior. The selection of specific antibody combinations and the selection of specific mRNA markers Made possible it but down to 10 cells per milliliter of blood without any non-specific background to capture the corresponding sought tumor cells. The background expression was eliminated and the sensitivity can be increased considerably (s. Band for Claudin 7).

3 shows the result of a method according to the invention with inoculated in blood tumor cells is shown from a Hodenkrebszellinie. In turn are selected from the blood sample all tumor cells are marked by one of the antibodies shown. Subsequently, a total of four mRNS- markers (GCAP, GA 733.2, GRPR and HGMI-C) is investigated. Here, that are detected with the use of only one antibody as Ber-EP 4 by means of the marker HGMI-C only down to 10 cells per milliliter of blood, and no testicular cancer cells are detected with the use of the antibody MOC-31 at all is shown. The same is true for the antibody 8B6 which has only a low sensitivity.

The combination of the antibodies Ber-EP4 and 8B6 also leads to a lack of detection by means of the marker HGMI-C as well as the combination of the antibodies Ber-EP4 and MOC-31st An optimal detection result for all four markers studied results for testicular tumor cells with the use of three antibodies Ber-EP 4, MOC-31 and 8B6, where detected down to 2 cells per milliliter of blood securely over each of the markers. According to the invention with the two detection reactions two markers proved so reliable detection of a minimum number of cells while avoiding a background detection can be carried out when selecting two markers from those used for 3 markers.

Figure 4 shows the detection of colon cancer cells were inoculated into the blood of a healthy person. This shows immediately that the use of a combination of the antibodies Ber-EP4 and MOC-31 results with respect to the mRNA marker EGF-R in an improved sensitivity. The use of the two antibodies at the same time a detection sensitivity of 100 cells per milliliter of blood is achieved, while the sensitivity is using only one antibody at about 1000 cells per milliliter of blood.

5 shows an experiment in which, by means of an antibody combination of Ber-EP 4, and HMPV.2 GP1.4, the labeled with at least one of the antibodies

Cells were selected and were then examined on the mRNA marker GA 733.2, CA 15.3, Her 2 and claudin. 7 However, here the blood of healthy people was no tumor cell but added definable te amounts of epithelial cells. As is apparent from Figure 5 directly, only two of the markers indicate a positive result in a very high number of epithelial cells.

In Figure 6 tumor cells were of two different

Breast cancer (MCF-7, and SKBR-3) was added to a blood sample. As antibodies, the antibodies Ber- Ep 4, HMPV.2 and GP1.4 were used in combination. As can be seen directly, is 7 are each secured to the use of the four mRNA markers GA 733.2, CA 15.3, Her 2 and claudin down guaranteed to 10 cells of the individual cell lines per milliliter detection of breast cancer cells. A non-specific reaction in blood without breast cancer cells did not occur. However, the tumor markers are GA 733.2 and 15.3 CA for cell line 2 (SKBR-3) sensitive, during tumor marker Her 2 is more sensitive to the cell line 1 (MCF-7). As well as the individual subtypes of breast cancer cells based on the marker pattern occurred can then be distinguished from each other.

Also in Figure 7 (MCF-7) were inoculated in Figure 7A and Figure 7B SKBR 3 in breast cancer cells in blood of different cell lines. When antibodies for the selectivity of the cells from the blood sample tion the antibodies Ber-EP 4, and HMPV.2 GP1.4 were used in combination. It can be seen immediately that 15.3 Her 2 and claudin to 2 cells per 5 milliliters reacts when using a combination of mRNA markers GA 733.2, CA 7 in each case at least one of the markers to down is positive, without the blood without tumor cells a would provide expression background. Here again recognize markers on the four different mRNS- a differential response of the two cell lines.

However, for example, are located in a blood sample, both cell lines, a detection of both cell lines would be by the selected marker combination in any case, down from 2 cells per 5 milliliters of blood guaranteed, ie, the detection of breast cancer cells in the blood would be independent of the cell type of the breast cancer cell line with high sensitivity possible.

Figure 8 shows the detection of colon cancer cells were inoculated in blood. The selection of the cells with the two antibodies Ber-EP4 and MOC-31 was carried out. The molecular biological detection step was taken with the mRNA markers GA 733.2, CEA and EGF-R. Two tumor cells were bar in 5 milliliters of blood detectable.

in turn, Figure 9 shows a measurement with a combination of three antibodies Ber-EP 4, MOC-31 were and 8B6 as well as the mRNA markers GCAP / PLAP, GA 733.2, GRPR and HMGI-C inoculated on blood, in the testicle cancer cells.

With each of the molecular markers of this triple combination of antibodies for cell selection in the immunological selectivity achieved with the use tionierungsschritt the detection of down to 2 cells per 5 milliliters.

Figure 10 finally shows the separation of selte- NEN cells from the cell mixture of a Biopsiemateri- as. To this end, biopsy material were obtained from breast tissue containing the tumor tissue with a suspected primary tumor, mechanically isolated and gauze of cell debris, connective tissue, etc. separately. The cell mixture obtained containing both cells of the suspected tumor tissue and cells of the surrounding healthy tissue was transferred to a cell selection having an input coupled to a solid phase antibody mixture (magnetic particles with antibodies GP1.4, HMPV.2 and Ber-EP 4) and after incubation magnetically separated for the preparation of the antigen-antibody binding. Subsequently, a mRNA was carried out with respect to the detection marker GA 733.2 and Her 2. As a control, a cell line of breast cancer was also determined at the same time in parallel. As can be seen, then a positive proof that the biopsy actually contained a breast cancer tumor.

The bands are marked in Figures 8 and 9 with "positive control" show the results of samples with the cell lines HT 29 for intestinal tumor (FIG. 8) and Tera / 1 for testicular cancer (Fig. 9).

Claims

claims
1. A process for selectioning and / or for the qualitative and / or quantitative detection of predetermined biological cells from or in a sample containing biological cells, characterized in that the sample having a predetermined combination of at least two antibodies and / or antibody derivatives, which with its binding sites preferably bind to different epitopes of about selektionierenden or cells to be detected, and / or with at least one bispecific antibody and / or antibody derivative, the / the to selektionierenden with its two binding sites to different epitopes of or cells to be detected preferentially binds, offset, with at least one of the antibodies and / or
Antibody derivatives labeled cells from the
wherein the at least two detection reagents with at least one ingredient to the selektionierenden or cells to be detected preferentially react sample are separated, and the separated cells are tested with a predetermined combination of at least two molecular-biological detection reagents.
2. Method according to the preceding claim, characterized in that at least two detection reagents are used to selektionierenden with various components of the cells to be detected and preferably react.
3. The method according to any one of the preceding claims, characterized in that the separation of the marked cells is effected in liquid phase or solid phase.
4. The method according to any one of the preceding claims, characterized in that coupled to solid-phase antibodies or antibody derivatives are used to separate the target cells from the sample.
5. The method according to any one of the preceding claims, characterized in that labeled with fluorophores antibodies or antibody derivatives are used and carried out the separation of the marked cells from the sample by means of flow cytometry (fluorescence-associated cell separation, FACS).
6. The method according to any one of the preceding claims, characterized in that are used with magnetic or pseudo-magnetic particles coupled antibodies or antibody derivatives and for the separation of the marked cells from the sample, the magnetic or pseudo-magnetic antibody-coupled particles after mixing with the sample magnetically by of the sample are separated.
7. The method according to any one of the preceding claims, characterized in that the antibodies or antibody derivatives have binding sites which bind preferentially to tumor cells.
8. The method according to any one of the preceding claims, characterized in that the antibodies or antibody derivatives
have binding Steep, the cells to one or more specific tumor types or - subtypes bind preferentially.
9. The method according to any one of the preceding claims, characterized in that for the separation of tumor cells or cells of a particular tumor type or subtype comprise the antibodies or antibody derivatives binding sites which bind to epitopes of an epithelial antigen, of an epithelial membrane antigen, of the antigen MUCL and / or antigen PLAP binding.
10. The method according to any one of the preceding claims, characterized in that at least one of the antibodies GP1.4, MOC-31, Ber-EP4, HMPV.2, 8B6, E29 and / or 131-11741 is used.
11. The method according to any one of the preceding claims, characterized in that, in general for the separation of tumor cells, or a specific type or subtype of a combination of antibodies is used, the antibody Ber-EP4 and MOC31 or at least two of the antibodies HMPV.2, GP1. 4 and Ber-EP4 contains.
12. The method according to any one of the preceding claims, characterized in that is used for the removal of breast tumor cells, a combination of antibodies that at least two of the antibodies 131-11741, GP1.4, E29 and HMPV.2 or at least two of the antibodies HMPV.2 containing GP1.4 and Ber-EP4.
13. The method according to any one of the preceding claims, characterized in that is used to separate colon tumor cells, a combination of antibodies containing antibodies Ber-EP4 and MOC-31st
14. A method according to any one of the preceding claims, characterized in that is used to separate testicular tumor cells, a combination of antibodies comprising at least two of the antibodies MOC31, Ber-EP4 and 8B6.
15. The method according to any one of the preceding claims, characterized in that as components of a sequence portion of a nucleic acid, DNA, RNA and / or sequence portions of different nucleic acids, DNA, RNA and / or different sequence portions thereof Nukleinsären, DNA, RNA and / or different alleles of the same nucleic acid, DNA, RNA, are detected.
16. The method according to any one of the preceding claims, characterized in that the separated cells are tested for the expression of a predetermined combination of at least two mRNA portions to the detection reagents, wherein selektionierenden to the expression of said at least two mRNA portions at least in the or detected cells is preferably carried out.
17. Method according to the preceding claim, characterized in that it is checked for the detection of tumor cells or cells of a particular tumor type or subtype, a combination of mRNA portions, mRNS- portions corresponding to sequence portions of at least two of the genes GA733.2, EGFR CEA, HER2 / neu, claudin-7 (CLDN7), GCAP (ALPPL2) / ALPP, GRPR, HMGIC, CK20, MAGE3, MUCL and stanniocalcin (STC1) contains.
18. Method according to the preceding claim, characterized in that it is checked for the detection of tumor cells or cells of a particular tumor type or subtype, a combination of mRNA portions mRNS- and portions corresponding to sequence portions of at least two of the genes EGFR, GA733.2 HER-2 / NEU contains.
19. The method of claim 16, characterized in that a combination of mRNA portions is used for the detection of breast tumor cells, the mRNA portions corresponding to sequence portions of at least two of the genes GA733.2, MUCL, Her-2 / neu, Claudin7, CK20, MAGE 3, stanniocalcin, EGFR and CEA contains.
20. The method of claim 16, characterized in that is used for the detection of breast tumor cells, a combination of mRNA portions mRNA portions corresponding to sequence portions of both genes GA733.2 and MUCL, corresponding to sequence portions of both genes Her-2 / neu and contains Claudin7 corresponding to sequence portions of at least two of the genes CK20, MAGE-3 and MUCL and / or corresponding to sequence portions of at least two of the genes stanniocalcin, EGFR and CEA.
21. The method according to claim 16, characterized in that is used to detect colon tumor cells, a combination of mRNS- sections containing mRNA portions corresponding to sequence portions of at least two of the genes CK20, EGFR, GA733.2, CEA and stanniocalcin.
22. The method according to claim 16, characterized in that is used to detect colon tumor cells, a combination of mRNS- portions mRNA portions corresponding to sequence portions of at least two of the genes CK20, EGFR, CEA and stanniocalcin and / or corresponding to sequence portions of at least two of the genes EGFR, CEA and GA733.2 contains.
23. The method according to claim 16, characterized in that is used to detect testicular tumor cells, a combination of mRNS- portions mRNA portions corresponding to sequence portions of at least two of the genes ALPP / ALPPL2 (GCAP), GA733.2 (= EGP-40 ), HMGI-C, GRPR contains.
24. The method according to any one of claims 16 to 23, characterized in that the mRNA portions are using polymerase chain reaction (PCR), LCR, NASBA RT-PCR and / or hybridisation techniques reproduced and / or detected.
25. The method according to any one of claims 16 to 24, characterized in that the mRNA of the separated cells is transcribed reversely into cDNA, the cDNA reproduced and then the presence or absence of the detected mRNA section is detected.
26. Method according to the preceding claim, characterized in that the amplified cDNA was digested by appropriate restriction enzymes and the presence or absence of the detected mRNA is detected based on the generated cDNA fragments (fragment analysis).
27. The method according to any one of the preceding claims, characterized in that the corresponding one of the detected mRNA cDNA is determined by fluorescence-based real-time PCR.
28. The method according to any one of claims 16 to 27, characterized in that the mRNA of the protein beta-actin is detected as internal control.
29. A diagnostic kit for selectioning and / or for the qualitative and / or quantitative detection of predetermined biological cells from or in a sample containing biological cells, characterized in that it contains a predetermined combination of at least two antibodies and / or antibody derivatives, which with their binding sites preferably bind to different epitopes of about selektionierenden or cells to be detected, and / or with at least one bispecific antibody and / or antibody derivative, the / the to selektionierenden with its two binding Steep to different epitopes of or cells to be detected binds preferentially and at least two molecular-biological
Detection reagents, wherein the at least two molecular-biological
Detection reagents with at least one
Part of the cells to be detected or to selektionierenden preferably react.
30. A diagnostic kit according to the preceding claim, characterized in that the at least two detection reagents preferably react with various components of the selektionierenden to or cells to be detected.
31. A diagnostic kit according to claim 29 or 30, characterized in that the antibodies or antibody derivatives marked with fluorophores.
32. A diagnostic kit according to any one of claims 29 to 31, characterized in that the antibodies or antibody derivatives coupled to solid phases,
33. A diagnostic kit according to the preceding claim, characterized in that the antibodies or antibody derivatives coupled to magnetic or pseudo-magnetic particles.
34. A diagnostic kit according to any one of claims 29 to 33, characterized in that the antibodies or antibody derivatives have binding sites which bind preferentially to tumor cells.
35. A diagnostic kit according to any one of claims 29 to 34, characterized in that the antibodies or antibody derivatives have binding sites which bind preferentially to cells of one or more specific tumor types or subtypes.
36. A diagnostic kit according to any one of claims 29 to 35, characterized in that for the separation of tumor cells, or a specific type or subtype of tumor cells comprise the antibodies or antibody derivatives binding sites which bind to epitopes of an epithelial antigen, of an epithelial membrane antigen, of the antigen MUCL and / or bind the antigen PLAP.
37. A diagnostic kit according to any one of claims 29 to 36, characterized in that it comprises at least one of the antibodies GP1.4, MOC-31, Ber-EP4, HMPV.2, 8B6, E29 and / or 131-11741.
38. A diagnostic kit according to any one of claims 29 to 37, characterized in that for the separation of tumor cells or cells of a particular tumor type or subtype, the antibody Ber-EP4 and MOC31 or at least two of the antibodies HMPV.2, GP1.4 and Ber- includes EP4.
39. A diagnostic kit according to any one of claims 29 to 38, characterized in that for the removal of breast tumor cells at least two of the antibodies 131-11741, GP1.4, E29 and HMPV.2 or at least two of the antibodies HMPV.2, GP1.4 and Ber-EP4 contains.
40. A diagnostic kit according to any one of claims 29 to 39, characterized in that it contains for the separation of colon tumor cells, the antibody Ber-EP4 and MOC-31st
41. A diagnostic kit according to any one of claims 29 to 40, characterized in that it contains for the separation of testicle tumor cells at least two of the antibodies MOC31, Ber-EP4 and 8B6.
42. A diagnostic kit according to any one of claims 29 to 41, characterized in that the detection reagents for the detection of a sequence portion of a nucleic acid, DNA, RNA and / or by sequence portions of different nucleic acids, DNA, RNA and / or of different sequence portions thereof Nukleinsären, DNS, RNS and / or are detected by different alleles of the same nucleic acid, DNA, RNA.
43. A diagnostic kit according to any one of claims 29 to 42, characterized in that the detection reagents are suitable for detecting the expression of a predetermined combination of at least two mRNA portions, the expression of these at least two mRNA portions at least in the cells to be detected or to selektionierenden preferably takes place.
44. A diagnostic kit according to any one of claims 29 to 43, characterized in that the detection reagents are suitable for detecting the expression of a combination of mRNA portions mRNA portions corresponding to sequence portions of at least two of the genes GA733.2, EGFR, CEA, HER2 / neu, claudin-7 (CLDN7), ALPPL2 (GCAP) / ALPP (PLAP), GRPR, HMGIC, CK20, MAGE-3, MUCL and stanniocalcin (STC1) contains.
45. A diagnostic kit according to any one of claims 29 to 44, characterized in that the detection reagents are suitable for detecting the expression of a combination of mRNA portions mRNA portions corresponding to sequence portions of at least two of the genes EGFR, HER-2 and GA733.2 / NEW contains.
46. ​​A diagnostic kit according to any one of claims 29 to 45, characterized in that the detection reagents for the detection of breast tumor cells are suitable for detecting the expression of a combination of mRNA portions corresponding to sequence portions mRNS- sections at least two of the genes GA733.2, Her -2 / neu, contains Claudin7, CK20, MAGE-3, MUCL, stanniocalcin, EGFR and CEA.
47. A diagnostic kit according to any one of claims 29 to 46, characterized in that the detection reagents are suitable for detecting the expression of a combination of mRNA portions for the detection of breast tumor cells that mRNS- portions corresponding to sequence portions of both genes and GA733.2 MUCL, corresponding to sequence portions of both genes contains Her-2 / new and Claudin7 corresponding to sequence portions of at least two of the genes CK20, MAGE-3 and MUCL and / or corresponding to sequence portions of at least two of the genes stanniocalcin, EGFR and CEA.
48. A diagnostic kit according to any one of claims 29 to 47, characterized in that the detection reagents for the detection of colon tumor cells are suitable for detecting the expression of a combination of mRNA portions mRNS- portions corresponding to sequence portions of at least two of the gene products CK20, EGFR, GA733 .2, CEA and stanniocalcin contains.
49. A diagnostic kit according to any one of claims 29 to 48, characterized in that the detection reagents for the detection of colon tumor cells are suitable for detecting the expression of a combination of mRNA portions mRNS- portions corresponding to sequence portions of at least two of the genes CK20, EGFR, CEA contains and stanniocalcin and / or corresponding to sequence portions of at least two of the genes EGFR, CEA and GA733.2.
50. A diagnostic kit according to any one of claims 29 to 49, characterized in that the detection reagents are suitable for detecting the expression of a combination of mRNA portions to detect testicular tumor cells mRNS- portions corresponding to sequence portions of at least two of the genes ALPP / ALPPL2 (GCAP ) GA733.2 (= EGP-40), HMGI-C and contains GRPR.
51. A diagnostic kit according to any one of claims 29 to 50, characterized in that the Nachweisreagenien for detecting the expression of DNA segments having at least two pairs of oligonucleotides, each oligonucleotide pairs is suitable as a primer pair for amplification of the to a respective one of mRNS- portions complementary cDNA.
52. A diagnostic kit according to the preceding claim, characterized in that at least one oligonucleotide is at least a pair of primers labeled with a fluorophore.
53. A diagnostic kit according to any one of the preceding claims, characterized in that for the detection of tumor cells or cells of a particular tumor type or subtype, the primer pairs for amplification of cDNS- portions corresponding to sequence portions of at least two of the genes GA733.2, EGFR, CEA, HER 2 / neu, claudin-7, ALPPL2 (GCAP) / ALPP, GRPR, HMGI-C, are CK20, MAGE3, MUCL and stanniocalcin suitable.
54. A diagnostic kit according to any one of claims 29 to 53, characterized in that it contains a reverse transcriptase, a polymerase, a Taq polymerase and / or restriction enzymes.
55. Use of a method or a diagnostic kit according to any one of the preceding claims for detecting cells rare type in suspensions and cell mixtures, in particular tumor cells, epithelial cells and / or endothelial cells in Kδrperflüssigkeiten, peripheral blood, sputum, ascites, lymph, urine, bone marrow and / or biopsy material and / or fetal cells in amniotic fluid or maternal peripheral blood.
56. Use according to the preceding claim for diagnostics and / or treatment control of tumor diseases.
57. Use according to the preceding claim for diagnostics and / or treatment control of testicular tumor, breast tumor and / or colon tumor.
PCT/EP2002/005489 2001-09-06 2002-05-17 Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells WO2003023057A2 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
DE10143691 2001-09-06
DE2001143775 DE10143775A1 (en) 2001-09-06 2001-09-06 Selection and determination of specific cells, useful particularly for diagnosis and monitoring of tumors, by antibody-mediated selection then detecting specific mRNA
DE10143699 2001-09-06
DE10143691.2 2001-09-06
DE2001143776 DE10143776A1 (en) 2001-09-06 2001-09-06 Selection and determination of specific cells, useful particularly for diagnosis and monitoring of tumors, by antibody-mediated selection then detecting specific mRNA
DE10143776.5 2001-09-06
DE10143775.7 2001-09-06
DE10143699.8 2001-09-06
PCT/EP2001/013606 WO2003044224A1 (en) 2001-11-22 2001-11-22 Diagnosis kit, dna chip, and methods for diagnosing or supervising the treatment of testicular cancer
EPPCT/EP01/13606 2001-11-22

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JP2003527120A JP4336198B2 (en) 2001-09-06 2002-05-17 Methods and diagnostic kits for cell selection and / or qualitative and / or quantitative detection
CA 2466896 CA2466896A1 (en) 2001-09-06 2002-05-17 Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells
US10/488,729 US7507528B2 (en) 2001-09-06 2002-05-17 Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells
EP20020732726 EP1409727B1 (en) 2001-09-06 2002-05-17 Method for qualitative and/or quantitative detection of cells
ES02732726T ES2253533T3 (en) 2001-09-06 2002-05-17 Procedure for qualitative and / or quantitative detection of cells.
AT02732726T AT309393T (en) 2001-09-06 2002-05-17 Process for the qualitative and / or quantitative detection of cells
DE2002504883 DE50204883D1 (en) 2001-09-06 2002-05-17 Process for the qualitative and / or quantitative detection of cells
AU2002304640A AU2002304640A1 (en) 2001-09-06 2002-05-17 Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells
ES02772273T ES2284927T3 (en) 2001-09-06 2002-09-06 Procedure and kit for diagnosis or control of cancer treatment.
DE2002509932 DE50209932D1 (en) 2001-09-06 2002-09-06 Method and kit for the diagnostic or treatment control of darm cancer
US10/488,828 US20050014208A1 (en) 2001-09-06 2002-09-06 Method and kit for diagnosing or controlling the treatment of breast cancer
EP20020774579 EP1409746A2 (en) 2001-09-06 2002-09-06 Method and kit for diagnosing or controlling the treatment of breast cancer
PCT/EP2002/009983 WO2003023059A2 (en) 2001-09-06 2002-09-06 Method and kit for the diagnosis or treatment control of intestinal carcinoma
EP20020772273 EP1409745B1 (en) 2001-09-06 2002-09-06 Method and kit for the diagnosis or treatment control of intestinal carcinoma
PCT/EP2002/009999 WO2003023060A2 (en) 2001-09-06 2002-09-06 Method and kit for diagnosing or controlling the treatment of breast cancer
AT02772273T AT359377T (en) 2001-09-06 2002-09-06 Method and kit for the diagnostic or treatment control of darm cancer

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